Diphtheria toxin (DT) is a protein exotoxin that is synthesized and secreted by Corynebacterium diphtheriae. The toxigenic strains of Corynebacterium diphtheriae contain a bacteriophage lysogen carrying the toxin gene. Mature form of DT is synthesized as a 535 amino-acid containing single polypeptide, which is derived from an initial 536 pro-peptide which undergoes proteolysis at positions 190, 192 and 193 to form the mature toxin. This splicing or proteolysis results into two subunits, A and B which are joined together by a disulfide bridge (Moskang et al Biol. Chem. 264: 15709-15713, 1989). Subunit A is catalytically active NAD-dependent ADP-ribosyl transferase portion. It is responsible for rendering Elongation Factor-2 (EF-2) inactive, and hence down regulates the protein synthesis in a target cell.
Diphtheria toxin is highly cytotoxic; a single molecule can be lethal to a cell, and a dose of 10 ng/kg can kill animals and humans. In course of providing an artificial immunity by injection of this toxoid, a process of detoxification should be included to render it safe for consumption by the recipient. Conventionally it was detoxified by chemical modification of the natural forms of DT, in such a manner that it still retains the required antigenicity required in a vaccine preparation.
Subsequently, a genetically detoxified form of Diphtheria Toxin known as Cross Reacting Material 197 or CRM197 was introduced; which essentially retains the immunological cross-reacting properties of DT.
CRM197 has been used in preparation of conjugate vaccines including Corynebacterium diphtheria, Hepatitis B, Bordetella pertussis, Clostridium tetani, Neisseria meningitides, Streptococcus pneumonia, Haemophilus influenza. It was generated by nitrosoguanidine mutagenesis of the toxigenic corynephage β, which was then used to infect Corynebacterium diphtheria. (Uchida et al Nature New Biology (1971) 233; 8-11, Nucleic Acids Res. 1984 May 25; 12(10):4063-9)
CRM197 has been studied for its potential use as a DT booster or vaccine antigen. The CRM197 protein has the same molecular weight as DT; but differs in a single base change in the A subunit i.e. a base change in the polynucleotide sequence of wild type DT, wherein a replacement of Guanine to Adenine results into an amino acid substitution at position 52, resulting into glutamic acid in CRM197 instead of glycine (Giannini G. et al., 1984). This point mutation results in a significant loss of toxicity and renders CRM197 safe for human use.
Production of significant quantities of diphtheria toxins such as CRM197 for use in vaccines has been hindered due to low level of expression in wild type bacteria. This problem has been addressed previously by expressing CRM197 in Escherichia coli by Bishai et al., (J. Baeteriol. 189:5140-5151). who describe the expression of a recombinant fusion protein containing diphtheria toxin (including the tox signal sequence), but this led to the production of degraded protein. The low yield in active form is also associated with degradation, improper folding, or both, depending on the specific characteristics, e.g., size and secondary structure, of the toxin. Hence as with most biopharmaceuticals, there is additional loss of expressed protein that occurs during the purification steps of CRM197, whereby maintaining a biologically active form of CRM197 poses a challenge. Therefore, there is need to achieve high level expression of bacterial toxoid CRM197 in an active form.
WO 2011/042516 discloses an improved process for making a bacterial toxin by periplasmic expression comprising the steps of a) growing a culture of the bacterial host cell containing an expression vector in which particular signal sequence are linked to the sequence of a bacterial toxin and b) inducing expression of the polypeptide containing particular signal sequence linked to a bacterial toxin such that a bacterial toxin is expressed periplasmically.
WO 2013/178974 A1 discloses a process for the intracellular expression of CRM197 in an Escherichia coli host, comprising expressing a vector comprising a gene encoding CRM197 operably linked to a Promoter and at feast one perfect Palindrome Operator sequence.
WO 2015/134402 A1 discloses a process for producing a recombinant CRM197 in a reduced genome of Escherichia coli host comprising incubating a reduced genome Escherichia coli comprising an expression vector comprising a nucleotide sequence encoding a CRM197 protein fused to a signal sequence that directs transfer of the CRM197 protein to the periplasm operably linked to an expression control sequence under conditions suitable for the expression of the recombinant CRM197 protein, whereby a yield of at least 1 gram per liter of soluble CRM197 is obtained and wherein the native parent Escherichia coli strain is a 12 strain, preferably K12 MG1655.
US 2012/0128727 A1 discloses an isolated nucleic acid molecule which encodes polypeptide CRM197, an expression vector comprising the isolated nucleic acid molecule and a method for recombinant production of a CRM197 tag fusion protein, comprising culturing the recombinant cell under conditions favoring production of said CRM197 tag fusion protein, and isolating said fusion protein.
US 2012/0289688 A1 discloses a process for periplasmic expression of a recombinant polypeptide by (A) Growing a culture of a gram-negative host cell; and (B) Inducing expression of a polypeptide such that a protein is expressed periplasmically; wherein one or more of the following steps is actioned during expression: (i) The pH of step a) is lower than the pH of step b); (ii). The temperature of step a) is higher than the temperature of step b); or (iii). The substrate feed rate of step a) is higher than the substrate feed rate of step b).
US 2014/0050758 A1 discloses a process for periplasmic expression of a bacterial toxoid comprising the steps of: a) growing a culture of a gram negative host cell in a fermentation medium, wherein the host cell is transformed with a polynucleotide, and wherein the polynucleotide encodes the bacterial toxoid and a periplasmic signal sequence; inducing expression of the bacterial toxoid;    b) maturing the host cell, wherein the maturing step comprises: I) subjecting the host cell to a pH shock: II) incubating the host cell with no feed addition; or III) subjecting the host cell to a temperature below −20° C.; and    c) extracting the bacterial toxoid from the host cell wherein the extraction process comprises osmotic shock wherein the gram negative host ceil is selected from the group consisting of Escherichia coli, Pseudomonas and Moraxella, wherein the host cell is alive during step b) and wherein the process is carried out in a fermenter which contains 10-5000 liters of culture.
U.S. Pat. No. 8,530,171 discloses a method for producing a recombinant toxin protein in a Pseudomonas host cell, said method comprising: ligating into an expression vector a nucleotide sequence encoding the toxin protein; transforming the Pseudomonas host cell with the expression vector; and culturing the transformed Pseudomonas host cell in a culture media suitable for the expression of the recombinant toxin protein; wherein the recombinant carrier protein is CRM197, and wherein the recombinant protein is produced at a yield of soluble or active CRM197 protein of about 0.2 grams per liter to about 12 grams per liter.
Conjugated polysaccharide vaccines that use CRM197 as a carrier protein have been approved for human use. These include: MENVEO® (Meningococcal (Groups A, C, Y, and W-135) Oligosaccharide Diphtheria CRM197 Conjugate Vaccine) (Novartis Vaccines and Diagnostics), a vaccine indicated for preventing invasive meningococcal disease caused by Neisseria meningitidis subgroups A, C, Y, and W-135; MENJUGATE® (Meningococcal Group C-CRM197 Conjugate Vaccine) (Novartis Vaccines and Diagnostics), a meningococcal group C conjugate vaccine; and PREVNAR® (Pneumococcal 7-valent Conjugate Vaccine (Diphtheria CRM197 Protein)) (Wyeth Pharmaceuticals, Inc.), a childhood pneumonia vaccine that targets thirteen serotypes of Streptococcus pneumoniae, and HIBTITER® (Haemophilus b Conjugate Vaccine (Diphtheria CRM197 Protein Conjugate)) (Wyeth), a Haemophilus influenzae type b vaccine. In addition, CRM197 has potential use as a boosting antigen for C. diphtheria vaccination and is being investigated as a carrier protein for use in other vaccines.
There has recently been a growing interest in CRM197 because of its potential antitumor action relating to its capacity to bind the soluble form of HB-EGF (Mekada et al, US Patent Publication NO. 2006/0270600A1). This antitumor function is attributable not only to CRM197, but also to other non-toxic derivatives of the DT toxin (e.g. the double mutant DT52E148K, or the fusion protein GST-DT). These mutants have been constructed by PGR, starting from the gene encoding CRM197. In said studies, however, the whole CRM197 was produced using cultures of C. diphtheria, grown at 35° C. for 16-17 hours. The CRMw was purified from the supernatant by means of an initial precipitation with ammonium sulphate, followed by three successive steps in ion exchange and hydrophobic chromatography (Mekada et al.).
Hence, there is an evident need for an alternative method for the production of CRM197 with high yield and cost-effective manner. Therefore, a method for economically producing CRM197 would greatly facilitate vaccine research, development and manufacturing.